Mutations in rsmG, encoding a 16S rRNA methyltransferase, result in low-level streptomycin resistance and antibiotic overproduction in Streptomyces coelicolor A3(2)
Mutations in rsmG, encoding a 16S rRNA methyltransferase, result in low-level streptomycin resistance and antibiotic overproduction in Streptomyces coelicolor A3(2)
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DOI：
10.1128/jb.01776-06
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发表时间：
2007-05-01
影响因子：
3.2
通讯作者：
Ochi, Kozo
中科院分区：
文献类型：
--
作者：
Nishimura, Kenji;Hosaka, Takeshi;Ochi, Kozo
导致高或低水平链霉素耐药性的某些str突变导致链霉菌过量生产抗生素。导致高水平耐药性的str突变发生在编码核糖体蛋白S12的rpsL中，而导致低水平耐药性的str突变尚不清楚。我们使用比较基因组测序来确定低水平耐药性是由rsmG突变引起的，rsmG编码一种s -腺苷甲硫氨酸（SAM）依赖的16S rRNA甲基转移酶，该酶含有SAM结合基序。野生型链霉菌中rsmG基因的缺失导致其对链霉素产生耐药性，并产生过量的放线菌素。在缺失突变体中引入野生型rsmG完全消除了rsmG缺失的影响，证实了rsmG突变是观察到的表型的基础。与早期使用自发性rsmG突变体的研究一致，携带Delta rsmG的菌株表现出SAM合成酶活性增加，这介导了抗生素的过量产生。此外，高效液相色谱分析显示，Delta rsmG突变体在16S rRNA上缺少一个7-甲基鸟苷修饰（可能在G518位置，对应于大肠杆菌的G527）。与某些rpsL突变体一样，Delta rsmG突变体在生长后期表现出增强的蛋白质合成活性。然而，与rpsL突变体不同，Delta rsmG突变体既没有表现出更高的70S核糖体复合物的稳定性，也没有增加核糖体再循环因子的表达，这表明rsmG和rpsL突变体增加蛋白质合成的机制不同。最后，自发性rsmG突变的发生频率是rpsL突变的1000倍。这些发现为研究rRNA修饰在链霉菌中激活次生代谢中的作用提供了新的视角。
Certain str mutations that confer high- or low-level streptomycin resistance result in the overproduction of antibiotics by Streptomyces spp. The str mutations that confer the high-level resistance occur within rpsL, which encodes the ribosomal protein S12, while those that cause low-level resistance are not as well known. We have used comparative genome sequencing to determine that low-level resistance is caused by mutations of rsmG, which encodes an S-adenosylmethionine (SAM)-dependent 16S rRNA methyltransferase containing a SAM binding motif. Deletion of rsmG from wild-type Streptomyces coelicolor resulted in the acquisition of streptomycin resistance and the overproduction of the antibiotic actinorhodin. Introduction of wild-type rsmG into the deletion mutant completely abrogated the effects of the rsmG deletion, confirming that rsmG mutation underlies the observed phenotype. Consistent with earlier work using a spontaneous rsmG mutant, the strain carrying Delta rsmG exhibited increased SAM synthetase activity, which mediated the overproduction of antibiotic. Moreover, high-performance liquid chromatography analysis showed that the Delta rsmG mutant lacked a 7-methylguanosine modification in the 16S rRNA (possibly at position G518, which corresponds to G527 of Escherichia coli). Like certain rpsL mutants, the Delta rsmG mutant exhibited enhanced protein synthetic activity during the late growth phase. Unlike rpsL mutants, however, the Delta rsmG mutant showed neither greater stability of the 70S ribosomal complex nor increased expression of ribosome recycling factor, suggesting that the mechanism underlying increased protein synthesis differs in the rsmG and the rpsL mutants. Finally, spontaneous rsmG mutations arose at a 1,000-fold-higher frequency than rpsL mutations. These findings provide new insight into the role of rRNA modification in activating secondary metabolism in Streptomyces.